We have discovered 2 ubiquitin sorting receptors (Ub-sorting receptors) that sort ubiquitinated membrane proteins (Ub-cargo) for degradation in the lysosome. One is the GGA coat protein, which appears to bind Ubcargo at the TGN and incorporate it into vesicles targeted for delivery to the endosome. The other is a complex between Vps27 and Hse1 (aka Mrs and STAM1/2 in animal cells), which recognizes Ub-cargo at the endosome and promotes its entry into luminal vesicles of multivesicular endosomes (MVB). This proposal aims to understand how these receptors work in relation to other putative Ub-sorting receptors and how they coordinate the activity of Ub ligases and Ub-peptidases that may alter the ubiquitination status of cargo. We will examine the molecular mechanisms that control the activity of this machinery, in large part by analyzing the function of various interactions between this machinery and other proteins. The Vps27-Hse1 complex associates with Ub ligases and peptidases, clathrin, Coatamer and other proteins, as well as the ESCRT-I complex that also binds Ub and participates in MVB biogenesis. We will determine what the functional roles of these interactions are and how the Vps27-Hse1 Ub-sorting function of Vps27- Hse1 is coordinated with other putative Ub-sorting complexes is such as ESCRT-I. We will also determine what other proteins work with Gga proteins to effect Ub-dependent sorting at the TGN by testing the potential of a variety of candidate proteins and conducting genetic screens to identify genes that operate in this pathway.